Part I: The Staining Process

Chapter 1

Introduction to
Immunohistochemistry
Clive R. Taylor, MD, D.Phil

Immunohistochemistry (n.)
Microscopic localization of specific antigens in tissues by staining
with antibodies labeled with fluorescent or pigmented material.
The American Heritage Medical Dictionary

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Introduction to Immunohistochemistry | Chapter 1

Chapter 1.1 Introduction

tochemical staining result start in the surgery operating room

and end at the interpretation of the stain by the pathologist,

Immunohistochemistry (IHC) is a method used to determine

which ultimately leads to treatment decision by the oncologist.

the expression of biomarkers in tissue. This educational guidebook will describe immunohistochemistry as it is used in the

For those new to the world of immunohistochemistry here

pathology laboratory as an aid in the differential diagnosis

is a brief outline of the steps needed to localize antigens in

and classification of cancer, and for certain other diseases,

tissues using antibodies for cancer diagnosis:

including infections. The factors that influence the immunohis-

Grossing
Accessioning
Tissue
processing &
embedding

Biopsy

Sectioning

Reporting

Screening,
interpretation
& archive

Staining
Enzyme

Secondary antibody

Dextran Backbone

Primary antibody
Tissue antigen
DAB
STEP C

STEP D

STEP E

Figure 1.1 From biopsy to reporting.

11

Chapter 1 | Introduction to Immunohistochemistry

Pre-Analytical Steps

Post-Analytical Steps

1. A Biopsy (surgically removed tissue specimen or needle

7. In the post-analytical process, the pathologist interprets the

biopsy) from the surgery room arrives in fixative at the

stains in context with positive and negative tissue controls,

pathology laboratory.

using bright field microscopy.

2. In the Accessioning room the sample details are entered

8. The results are reported to the oncologist for treatment

into the laboratory information system (LIS). A barcoded

decision.

label can ensure track and trace capabilities.

3. During Grossing, the specimen is visually examined for

Processing
Decalcification

suspicious areas that require further examination. Samples

from the specimen that require further microscopic testing are

excised as tissue blocks and placed in barcoded cassettes.

4. Tissue processing and embedding are the steps where

the tissue block is processed into a form and condition suitable

for making ultrathin microscopic sections. Typically, the tissue

is fixed in formalin then dehydrated before it is embedded

in paraffin.

5. Sectioning is the fine art of cutting the paraffin-embedded

tissue blocks into ultrathin (~4 m) sections and placing them

onto glass slides. A barcode on the slide can ensure trace-

Embedding
Vax, type, storage

Pre-analytic

requested test for that particular section.

Platform
Manual, automated

passes antigen retrieval, application of the primary antibody

and visualization system, ending with counterstaining:

a. Antigen retrieval is performed to recover the antigens that

Analytic
Visualization system
Sensitivity, specificity
enhancement

Primary antibody
Clone, dilution
buffer, time, temp
Chromogen
Sensitivity, localization

may have been altered by fixation;

Counterstain
Time, color

Pre-treatment
Proteolysis, HIER,
time, temp, pH

Analytical Steps
6. Staining is the analytical part of the IHC process. It encom-

Sections
Thickness, drying,
storage

Fixation
Delay, time,
type, volume

ability and may also contain protocol information for the

Tissue
Type, dimensions,
biological variation
cauterization

b. Endogenous enzymes are blocked (this step can also be

performed after primary antibody incubation);

c. A primary antibody is applied that specifically binds to

the antigen of interest;

d. The secondary antibody carries the label (enzyme); upon

application it binds to the primary antibody;

e. Chromogen is applied to visualize the antibody/antigen

Interpretation
Quantification,
localization,
pos./neg. def.
cut-off level,
panels, algorithms

Post-analytic

Control
Internal/external,
critical stain quality
indicators

complex;
f. Counterstaining is performed to visualize nuclei and overall
tissue architecture;

Reporting
Diagnostic context

g. Sections are dehydrated, mounted and coverslipped.

Figure 1.2 Many factors may influence the IHC staining result. With just
3 choices at each of 14 steps there are 4.8 million different procedures!

12

Introduction to Immunohistochemistry | Chapter 1

Chapter 1.2 History of immunohistochemistry

cein and visualized by ultraviolet light (fluorescence microscopy)

(1). During the next 25 years, the Coons method was used with

This IHC Educational Guidebook will describe the potentials

different modifications, including labeling with heavy metals, but

and pitfalls in the immunohistochemical staining process from

it was not until the introduction of enzyme-labeled antibodies (2)

biopsy to interpretation, with special attention to the analytical

that the method overcame many of the inherent issues with fluo-

processes and how to improve certainty in the staining result by

rescein and heavy metal labeling of antibodies. In the early 1970s,

employing standardization to the processes.

application of the immunoperoxidase method to formalin paraffin

embedded tissues by Taylor, Mason and colleagues in Oxford,

Before immunohistochemistry reached its now widespread use

was a critical step in extending use of the method into routine

as an important method in routine cancer diagnosis, the tech-

diagnosis in anatomic pathology. The direct labeling method had

nology had a long history of technological developments out-

the drawback that each individual primary antibody, or the secon-

lined in the table below.

dary antibody, had to be labeled with enzyme. That problem was

circumvented by the development of an unlabeled antibody en-

Table 1.1 The major milestones in the history of immunohistochemistry.

zyme method, the peroxidase anti-peroxidase (PAP) method,

which had the further advantage of increased sensitivity, facili-

Year

Method

References

1941

Fluorescence-labeled primary antibodies

Coons et al (1)

1967

Enzyme-labeled primary antibodies

Nakane & Pierce (2)

1970

Secondary un-labeled antibodies (PAP)

Sternberger et al (3)

and improved detection systems for visualization of antigens in

1970

Detection of antigens on ultrathin

sections

Kawarai & Nakane (4)

tissue, IHC suffered from lack of reproducibility, due in part to poor

1974

Application to routine formalin

paraffin sections

Taylor et al (5-7)

verse effects of fixation.

1975

Invention of monoclonal antibodies

Khler & Milstein (8)

Increased demand led to better quality reagents from the com-

1978

Double staining using un-labeled

antibodies (APAAP)

Mason & Sammons (9)

mercial sector, with improved quality control of production

1979

Monoclonal antibodies to human

antigens

McMichael et al (10)

1988

Capillary gap semi-automated staining

Brigati et al (11)

1991

Heat-induced antigen retrieval

Shi et al (12)

Dr. Niels Harboe, founder of Dako, realized in the early 1970s

1993

Standardization efforts as Total Tests

Taylor (13)

the need for standardized antibody preparations for safe and

1995

Dextran-polymer-based detection system

Dako

reproducible diagnoses and began producing purified poly-

1998

Immunohistochemistry as companion
diagnostics

Dako (HER2)

2007

Recommendations for improved

standardization of IHC

Goldstein et al (14)
Wolff et al (15)

Even with the purified and highly specific polyclonal antibodies

2008

Molecular HER2 CISH Tests in the

IHC lab

Invitrogen

there was a need for improved specificity of antibodies and a

tating use in routine tissues. A related parallel development was

the introduction of the alkaline phosphatase anti-alkaline phosphatase (APAAP) in 1978 (9). Even with the development of new

quality antibody reagents, and in part to the inconsistent and ad-

methods. Polyclonal antibody preparations differ between

serum samples in affinity and specificity, as the immuneresponse changes with time and immunization preparations,
and as one animal is replaced by another as the source.

clonal antibodies that had the same strength (as measured by

titer) from batch to batch.

greater variety in terms of target proteins. The invention, in 1975,

of hybridomas that could produce monoclonal antibodies (8) resulted in the production of the first monoclonal antibody that was

Professor Albert H. Coons and co-workers demonstrated in 1941

highly specific for human thymocytes using hybridoma technolo-

that it was possible to localize antigens in tissue slices using an-

gy (10). Monoclonal antibodies paved the way for a rapid growth

tibodies against Streptococcus pneumoniae labeled with fluores-

in the use of IHC in research and diagnosis of cancer.

13

Chapter 1 | Introduction to Immunohistochemistry

One other consequence of the lack of reproducibility was the

development of automated instruments (11). Automation was

Label

invented with the fundamental thought that a properly functioning and maintained instrument will consistently perform its

Primary antibody

pre-programmed instructions in the same way slide after slide-

Tissue antigen

Direct method (one step)

The primary antibody (green)
is labeled with an enzyme
or fluorescence.

which is the principal reason why an instrument potentially can

give superior reproducibility, compared with manual methods.
However, progress was slow until 1991, when Shi et al (12) in-

Enzyme

troduced antigen retrieval (or heat-induced epitope retrieval),

thereby facilitating extension of IHC to a much broader range

Secondary antibody

of applications in formalin paraffin sections, but at the cost

of adding yet another variable to the process. This important
publication on antigen retrieval thus gave new insights and
impetus to efforts in standardization of IHC, leading to the intro-

Indirect method (two steps)

An enzyme-labeled secondary antibody reacts with unconjugated primary
antibody bound to tissue antigen.

Primary antibody
Tissue antigen

duction of the Total Test concept (13) as a result of a series of

meetings sponsored by the Biological Stain Commission and
the FDA in the early 1990s.
Peroxidase
anti-peroxidase complex

The standardization efforts, coupled with attempts to use IHC

in a semi-quantitative setting raised demands to a new level,
exemplified by the introduction, in 1998, of the HercepTest
(Dako), which was the first cancer companion diagnostic, in
this instance designed for selection of breast cancer patients

Secondary antibody

for treatment with the new drug Herceptin (Genentech/Roche). Clinical trials had shown that patients whose tumors over-

Primary antibody

expressed HER2 would benefit the most from Herceptin treat-

Tissue antigen

ment. The HercepTest assay uses IHC on patient samples,

Unlabeled method
Pre-formed enzyme immune
complex reacts with
secondary antibody.

in combination with control cell lines having known HER2 expression to determine if a breast cancer overexpresses HER2.
Some 15 years later, this assay together with similar HER2 assays from other vendors, still serves as a rare example of a

Dextran backbone
HRP enzyme

semi-quantitative IHC assay used in routine clinical pathology.

The polymer-based visualization system, introduced shortly
before HercepTest, is the most widely used detection method
in IHC today, with advantages of stability and high sensitivity.
The technical advances in IHC in the last decade have been

Primary antibody

incremental, with little impact on the basics of the method.

Tissue antigen

Automation has become more advanced, including laboratory

information system integration, with track and trace of samples,
while whole slide digital imaging is slowly being integrated into
the analysis of stain result. These advances can best be regarded
as improvements in standardization; a process that started back

14

Secondary antibody,
mouse/rabbit

Labeled polymer
A long dextran
polymer is labeled
with both the secondary
antibody and multiple
enzyme molecules.

Figure 1.3 The development of detection systems used for IHC.

Please see Chapter 6 for a full description of the many different detection methods.

Introduction to Immunohistochemistry | Chapter 1

in the early 1990s and was re-emphasized in the 2007 publications by Goldstein et al (14) and Wolff et al (15), but also by the
work being done e.g. estrogen receptor assessment (16, 17). The

Chapter 1.3 Standardization in Clinical

Immunochemistry vs.
Immunohistochemistry

critical importance of IHC standardization became evident with

the revelation of disturbingly high numbers of false negative or

For more than 30 years, clinical immunochemistry has em-

false positive results in IHC determinations of ER (estrogen recep-

ployed blood or urine samples to determine the concentra-

tor) and PR (progesterone receptor) expression, and also HER2.

tion of certain biomarkers, e.g. creatinine and cystatin C for

In one example, a re-testing in 2007 of 1,023 breast cancer sam-

evaluation of kidney function, and C-reactive protein as a

ples from Newfoundland revealed that approximately 1 out of 3

marker of inflammation. Although clinical immunochemis-

samples was scored falsely ER negative (17). As a consequence

try covers a multitude of assay types, most of these tests

of the false negative ER test results, these women were not ac-

are based on the ELISA (enzyme-linked immunosorbent as-

corded the potential benefit of anti-hormonal therapy.

say) method, a method that closely parallels IHC in principle. One major difference is that International Reference Materials and Calibrators are used in clinical immunochemistry

Immunohistochemistry publications

(ELISA) to achieve quantitative results from these assays.

140.000
122198
115006

120.000
100.000

Immunohistochemistry is based on principles similar to the

ELISA method, yet it is at best a semi-quantitative method for

94391

determination of the expression of biomarkers in tissue sam-

80.000

75352

60.000

ples. However, IHC should not be regarded as simply another special stain, like a PAS stain or a silver stain. IHC is es-

61315

40.000

sentially an ELISA method applied to a tissue section. In this

32626

respect, when correctly performed, IHC has the potential

2
01

-2
20

08

00

-2
20

03

00

7
19

98

-2

99

19

93

-1

99

-1
19

88

98

-1

98

83
19

-1

97

78
19

-1

97

73
19

-1

68
19

63
19

ELISA assay; much more than a simple stain. That the IHC

to perform as a reproducible and quantitative tissue-based

96

9044
3009 7423

-1

20.000

14319

Interval (Years)

method does not perform to this level, reflects deficiencies in

the application of the method, specifically inconsistent sample preparation, lack of reference or calibration standards,

Figure 1.4 The number of IHC publications in the last 50 years. The
data are from Pubmed using the search term immunohistochemistry.

and inadequate validation of reagents (18, 19). If ELISA can

use a standard curve to convert the measured immunoreactivity into a quantitative amount of tested protein, then IHC
in theory can also convert the IHC intensity observed in

The latest development in cancer diagnosis is the inclusion

FFPE tissue sections into the amount of tested protein by an

of molecular tests (FISH/CISH) in anatomic pathology labs,

equivalent standard ruler. Comparative studies of IHC inten-

driven by HER2 assessment requirements. Other techno-

sity on frozen tissue vs. FFPE tissue have shown identical

logies also are entering into the pathology lab and into rou-

intensity by using an optimized AR protocol (20, 21), and

tine diagnosis, and technologies such as array comparative

similar protein quality is evident when examined by mass

genomic hybridization or next generation sequencing will likely

spectrometry (22), leaving no theoretical reason for lack of

be a fundamental part of cancer diagnosis in the future. One

true quantitative IHC assays. Nonetheless, today IHC as-

ongoing goal is to interface these newer methods of molecu-

says are at best no more than semi-quantitative, for reasons

lar analysis with existing and improved morphologic criteria, a

that are more of a practical nature.

field termed Molecular Morphology.

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Chapter 1 | Introduction to Immunohistochemistry

Chapter 1.4 Growing Consensus

for Standardization

Table 1.2 The Total Test: An IHC stain should be managed in the same rigorous manner as a clinical laboratory analysis. Modified from Taylor (14, 24).

Pre-analytic

From the beginning there has been concern relating to the

reproducibility of immunohistochemical methods as applied

Test selection

to formalin-fixed, paraffin-embedded (FFPE) tissue sections.

Specimen type

A consequence of not controlling all parameters (in fixation,

Acquisition, pre-fixation/transport time

processing and staining) is poor day to day reproducibility within a single laboratory, and poor reproducibility among

Fixation, type and total time

Processing, temperature

different laboratories. In recent years these concerns have

increased and lack of standardization, well shown in inter-

Test selection

laboratory quality assurance surveys performed by NordiQC

Antigen retrieval procedure

and UK NEQAS, is now recognized as a major impediment

to basic research, clinical trials, and direct patient care. Over
the past three decades a number of conferences have been

Selection of primary antibodies

Protocol; labeling reagents

held to address this topic and to seek constructive resolutions.

Reagent validation

Among the most productive were a series of meetings spon-

Control selection

sored by the Biological Stain Commission and the FDA in the

Technician training/certification

early 1990s, that led to recommendations for manufacturers

concerning the precise description and validation of IHC rea-

Laboratory certification / QA programs

gents (23), and also highlighted the necessity to pay attention

Post-analytic

to all aspects of the IHC test procedure. The latter recommen-

Assessment of control performance

dation, borrowed from the much more rigorous protocols ap-

Description of results

plied to immunologic assays in clinical laboratories, became

known as the Total Test approach (Table 1) (23, 24). A decade later a meeting of the FDA and NIST (National Institute of

Interpretation/reporting
Pathologist, experience and CME specific to IHC

Standards and technology) focused upon standardization of

HER2 IHC assays, and the need for universal control materials

In the analytical steps, antigen retrieval is the first challenge.

(reference standards) (25).

Different antigens require different antigen retrieval for optimal

staining results, and the different variations of the AR process

Chapter 1.5 Standardization Starts

in the Surgery Room

add another variable that must be controlled. Antigen retrieval

is described in detail in Chapter 3.
Selecting the right antibody for the right marker is one of the

While Table 1.2 only mentions a few of the major steps in a To-

key steps in the analytical process. Some monoclonal anti-

tal Test, the pre-analytical process alone contains at least 62

body clones are more specific than others against the same

identifiable steps of which 27 have been examined in published

biomarker. In other cases a polyclonal antibody may be the

research. Out of these 27 steps, 15 pre-analytical variables are

best choice. Selection of the primary antibody is described in

capable of impacting the immunohistochemistry staining result

detail in Chapter 4.

including fixation delay, fixative type, time in fixative, reagents

16

and conditions of dehydration, clearing, paraffin impregnation

Using a protocol that is optimized for the detection of the bio-

and conditions of slide drying and storage (26). Pre-analytical

marker is vital. The optimal protocol must be able to identify

variables are described in detail in Chapter 2.

the antigen of interest in cells and structures with both low and

Introduction to Immunohistochemistry | Chapter 1

high expression. Optimization of the staining protocol is de-

Post-analytical standardization is essential for prognostic or pre-

scribed in detail in Chapter 5.

dictive biomarkers, e.g. HER2 and ER/PR, adhering to specified

stain interpretation guidelines to give the sample a scaled score

The final step of the analytical process is the visualization of the

(e.g. from 0-3+). However, most biomarkers are used for cell line-

antigen/antibody reaction. Here the selection of the detection

age and tissue identification, where expression levels are usually

system must consider the complexity of the visualization and the

not as critical and interpretation is not linked to a semi-quantita-

required amplification needed to visualize the biomarker. The var-

tive scoring system, but is reported as a binary Yes or No sys-

ious detection systems are described in detail in Chapter 6.

tem (positive or negative) for the tested biomarker. Digital analysis

of IHC stains is described in Chapter 7.

Chapter 1.6 Future Aspects for Standardization

of Immunohistochemistry

Table 1.3 Major steps affecting the immunohistochemistry staining result.

Step

Effect on IHC

Biopsy

Depending on the suspected cancer type, tissue samples

can be obtained in different ways such as punch/core
biopsy, excisional/incisional biopsy, etc. Tissue
degradation begins at the time of sample removal.

Fixation

Embedding

Sectioning
and Mounting

Antigen
Retrieval

Primary
Antibody

Visualization

Interpretation

The sample should be fixed as soon as possible after

surgery, ideally within less than an hour. The chemical
fixation crosslink proteins in the sample thereby
stopping the degradation process. Too short or too
long fixation can affect the staining result.

The consensus arising from the standardization efforts is that the

reliability and reproducibility of IHC methods in routine surgical
pathology have been greatly hindered by two key factors.
1. While reagents available for IHC have increased in quality,
there has been an even greater increase in number of

sources and variety of staining methods. This plentitude of

After fixation, the sample is embedded in paraffin

for long-term storage and to enable sectioning for
subsequent staining. Once embedded in paraffin,
samples can be stored (almost) indefinitely.

reagents contributes to lack of standardization in signifi-

Formalin-fixed, paraffin-embedded tissues are

sectioned into thin slices (4-5 m) with a microtome.
The sections are then mounted onto adhesive-coated
glass slides.

to requirements for such high standards of excellence in

Due to the fixation process, an antigen retrieval

treatment is applied to unmask the epitopes, either
by heat (heat-induced epitope retrieval; HIER) or
enzymatic degradation (proteolytic-induced epitope
retrieval; PIER). Incorrect antigen retrieval for the
biomarker of interest will adversely affect the
staining result.

cant ways, that in theory are manageable by good tech nique and use of proper controls, but in practice have led
the technical process, that many laboratories cannot find

sufficient, or sufficiently skilled, staff to comply.

2. The usual method of sample preparation for tissue remains

as formalin fixation and paraffin embedment (FFPE). This

venerable approach may be satisfactory for the preservation

of morphologic detail, but does adversely affect the antigenicity

of many target molecules in the tissue, to degrees that are

unknown. The enormous variation in protocols (including

An antibody with specificity for the biomarker of

interest is applied. The specificity and sensitivity of
the antibody affect the staining result.

fixation times) employed for FFPE among different laborato-

ries, or within the same laboratory from specimen to speci-

The antigen/antibody complex signal is amplified and

visualized using a detection system. The strength of
amplification of the reaction affects the staining result
(intensity).

The staining pattern is assessed by a pathologist in

context with other biomarkers, controls and other tests
(e.g. H&E, special stains. Inter- and intra-observer
variability is common, especially for semi-quantitative
assays. This variability highlights the importance of
training and inter-calibration.

men, compounds the problem and contributes to the

current poor reproducibility.

While several decades have passed, these issues have not

been satisfactorily addressed. Legions of investigators, and
many manufacturers, have addressed different aspects of
the problem, focusing upon better sample preparation (fixation), more effective methods of antigen retrieval, improved

17

Chapter 1 | Introduction to Immunohistochemistry

reagents, more sophisticated automated platforms, more sen-

high-quality reagents are available, with highly sensitive

sitive detection methods, and the development of reference

detection methods, but they must be employed pro-

standards or controls (13, 23-25).

perly in controlled fashion, and currently often are not.

In order to improve the quality and reproducibility from sample to

sample, and lab to lab, the accreditation process for many pa-

there is a pressing need for tissue-based IHC controls (or

thology laboratories now includes participation in external quality

reference standards) (19, 25) that can be made available

assurance (EQA) schemes. EQA organizations, like NordiQC, UK

to all laboratories performing IHC assays, somewhat ana-

NEQAS and CAP, are independent organizations not associated

logous to the international reference standards and cali-

with commercial suppliers. Their role is to promote the quality of

brators that are available to clinical laboratories performing

immunohistochemistry (and in situ hybridization) by arranging

ELISA testing.

Participation in EQA schemes can help laboratories

improve the reproducibility;

external QA schemes for pathology laboratories. Similar EQA

schemes are now available in many countries and regions around

From this brief discussion it follows that to improve standardi-

the world. The purpose of EQA schemes is to improve the quality

zation to the point that all laboratories would carry out the IHC

of staining results in the participating laboratories; thus it is the

in identical fashion for every phase of the Total Test; it would

individual labs that are being assessed. It is their choice of an-

require them to use the same fixative and fixation time (adjusted

tibody, visualization system, instrumentation and protocol that is

to tissue type), the same antigen retrieval process, the same pri-

the basis for the EQA organization's evaluation and feedback. A

mary antibodies and detection systems, with the same automat-

lab volunteers to participate in the assessment runs. Laboratories

ed stainer and common controls. Clearly this perfect option will

typically enroll for a year, during which they receive approximate-

never happen, and we therefore must do what we can to reduce

ly 16 unstained tissue slides (NordiQC), or 7-8 different modules,

the consequences of the variables in the process.

where each module usually has two tissue slides (UK NEQAS), to
stain using their own internal standard protocols for those markers

Ultimately the overriding factor in effecting significant change

designated by the QA organization. The labs return the stained

must be to transform the mindset of pathologists, at least of

slides to the QA organization for assessment, which is conducted

the next generation, to the view that the end result of an IHC

by experts engaged by the organization. The labs receive either

protocol is not just a stain, with intensity to be adjusted at the

a Passed rating or Not Passed rating. Both NordiQC and UK

whim of the pathologist. Rather IHC is a precise immunoassay

NEQAS inform all participants of their individual scores and pro-

that is strictly quantifiable, and must be performed only with a

vide suggestions for protocol optimization when required. Both or-

degree of technical rigor and control that matches any other

ganizations present the anonymous results on their web sites, with

immunologically-based assay of like principle (namely ELISA).

statistics and best method for the particular marker.

ELISA is a gold standard method for quantitative assays in the

clinical laboratory. ELISA reagents are purchased in prepared

CAP (College of American Pathologists) in the US, has a simi-

form, with all of the necessary reagents, defined protocols,

lar QA process, but requires only the return of stain results and

and reference or calibration standards, for use with specified

interpretation, not the stained slides.

instrumentation. Ready-to-use reagents, coupled with proven

detection systems, fixed and validated protocols, recommend-

18

Some broad conclusions are possible:

ed controls and automation, represent an analogous pathway

resolution of the problem of pre-analytical sample prep-

that could, if widely adopted with appropriate controls, lead to

improved levels of reliability and performance for IHC.

aration is not imminent; the practical aspects of developing

tissue handling and fixation procedures that fit the daily

routine of every hospital are challenging. Importantly the

logistical obstacles to implementation of standardized sam-

ple preparation procedures worldwide are formidable;

Introduction to Immunohistochemistry | Chapter 1

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3.

Sternberger LA, Hardy PH, Cuculis JJ, Meyer HG. The unlabeled
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